Vascular smooth muscles (VSMCs) play a major role in the normal and pathologic responses of the arterial wall to injury. VSMCs are normally present in the arterial tunica media where they regulate vascular tone and blood flow. In the vessel wall, VSMCs are surrounded and separated from other cells by extracellular matrix (ECM). Arterial injury leads to proliferation of medial VSMCs and migration of these cells from the media to the intima. These steps are dependent on the local degradation and remodeling of the ECM. Our previous studies in the rat carotid artery stenosis model indicated that the activation of matrix metalloproteinase 2 (MMP-2) was increased from 5-7 days after balloon injury during the time VSMCs migrated to the neointima. Therefore, in order to inhibit VSMCs migration from the media to the intima we constructed a replication-deficient adenovirus vector which carries the cDNA for human tissue inhibitor of metalloproteinase 2 (AdCMV.TIMP-2), a natural inhibitor of MMP-2. In vitro infection of VSMCs with this vector caused a significant overexpression of TIMP-2 as assessed by Western analysis. The conditioned media from infected cells inhibited VSMCs invasion in a Boyden chamber assay with Matrigel-coated filter while it did not have any effect on VSMCs migration alone in a similar assay with an uncoated filter. Subsequently, the rat carotid artery model of vascular injury was used to study the effect of in vivo infection of the arterial wall with an AdCMV.TIMP-2 on neointima formation. Preliminary experiments were aimed at establishing the optimal concentration of virus and timing for infection with an Ad vector expressing the beta-galactosidase (beta-gal) into balloon injured rat carorid arteries. Our results revealed that under our experimental conditions 50% to 70% of medial cells in the infected area showed positive beta-gal staining. In addition, analysis of beta-gal expression in these segments showed persistence of beta-gal staining up to 6 days after infection.